DNA methylation, or the covalent addition of a methyl group to cytosine within the context of the CpG dinucleotide, has profound effects on the mammalian genome. These effects include transcriptional repression via inhibition of transcription factor binding or the recruitment of methyl-binding proteins and their associated chromatin remodeling factors, X chromosome inactivation, imprinting and the suppression of parasitic DNA sequences. DNA methylation is also essential for proper embryonic development; however, its presence can add an additional burden to the genome. Normal methylation patterns are frequently disrupted in tumor cells with global hypomethylation accompanying region-specific hypermethylation. When these hypermethylation events occur within the promoter of a tumor suppressor gene they may silence the gene and provide the cell with a growth advantage in a manner akin to deletions or mutations. Furthermore, DNA methylation may be an important player in both DNA repair and genome stability.
DNA methylation at the 5-position of cytosine in CpG dinucleotides is an important aspect of physiological processes including embryonic development, X chromosome inactivation, imprinting, and transcriptional regulation. While CpG dinucleotides are generally methylated throughout the genome of normal somatic cells, CpG islands (CGIs), clusters of CpG dinucleotides in gene regulatory regions, are usually unmethylated. Aberrant hypermethylation of CGIs and subsequent transcriptional repression is one of the earliest and most common somatic genome alterations in multiple human cancers. Some cancers even seem to exhibit a so-called CpG island methylator phenotype (CIMP). The rapid and sensitive detection of DNA hypermethylation, therefore, would not only enhance our understanding of how DNA methylation may contribute to carcinogenesis, but could aid in early cancer diagnosis and risk stratification.
Most of the current DNA methylation detection strategies use sodium bisulfite to deaminate cytosine to uracil while leaving 5-methylcytosine intact (Wang et al., Nucleic Acids Res., 8:4777-90 (1980)). Among these, methylation specific PCR (MSP) (Herman et al., Proc. Natl. Acad. Sci. USA, 93:9821-26 (1996)) uses PCR primers targeting the bisulfite induced sequence changes to specifically amplify either methylated or unmethylated alleles. Quantitative variations of this technique, such as “MethyLight” (Eads et al., Nucleic Acids Res., 28:E32 (2000)), “HeavyMethyl” (Cottrell et al., Nucleic Acids Res., 32:e10 (2004)), and “MethylQuant” (Thomassin et al., Nucleic Acids Res., 32:e168 (2004)), employ methylation specific oligonucleotides in conjunction with Taqman probes or SYBR Green based real-time PCR amplification to quantify alleles with a specific pattern of methylation. These techniques are highly sensitive and specific for detection of DNA methylation. However, bisulfite based techniques are quite cumbersome, involving time- and labor-intensive chemical treatments that damage DNA and limit throughput. Additionally, PCR primer design becomes difficult due to the reduction in genome complexity after bisulfite treatment, leading to an inability to interrogate the methylation pattern at some or all CpG dinucleotides in a genomic locus of interest.
Other DNA methylation detection assays use methylation-sensitive restriction enzymes to digest unmethylated DNA while leaving methylated DNA intact for detection by Southern blot analysis (Singer et al., Science, 203: 1019-1021 (1979); Bird & Southern, J. Mol. Biol., 118:2747 (1978); Pollack et al., Proc. Natl. Acad. Sci. USA, 77:6463-67 (1980); Feinberg & Vogelstein, Nature, 301:89-92 (1983)), PCR (Singer-Sam et al., Mol Cell Biol., 10:4987-89 (1990); Singer-Sam et al., Nucleic Acids Res., 18:687 (1990)), or real-time PCR (Bastian et al, Clin. Cancer Res., 11:4037-43 (2005)). The Southern blot strategy is not easily amenable to high throughput analysis, and requires copious amounts of high molecular weight DNA. Digestion followed by PCR is sensitive, but is limited to interrogating methylation only at the enzyme recognition sites and is plagued by a propensity for false-positives resulting from incomplete digestion.
Another strategy for in vitro methylation detection, first introduced in 1994 by Cross et al. (Nat Genet, 6: 23644), uses column- or bead-immobilized recombinant methylated-CpG binding domain (MBD) polypeptides, particularly MECP2 (Cross et al, supra; Brock et al., Nucleic Acids Res., 29:E123 (2001); Shiraishi et al., Proc. Natl. Acad. Sci. USA, 96:2913-18 (1999)) and MBD2 (Rauch & Pfeifer, Lab. Invest., 85:1172-80 (2005)), to enrich for methylated DNA fragments for subsequent detection by Southern Blot, PCR, or microarray hybridization. The MBD proteins are thought to bind specifically to methylated chromosomal DNA in mammalian cells (Ballestar et al., EMBO J., 22:6335-45 (2003)), facilitating transcriptional silencing (Bakker et al., J. Biol. Chem., 277:22573-80 (2002); Lin et al., Cancer Res., 63:498-504 (2003)) by recruitment of chromatin remodeling and transcriptional repression complexes (Wade, Bioessays, 23:1131-37 (2001); Feng & Zhang, Genes Dev., 15:827-32 (2001)). A recent version of this strategy, called MIRA (Rauch & Pfeifer, supra), uses fill-length MBD2 immobilized on magnetic beads to enrich for methylated DNA with subsequent detection of candidate methylated genes by PCR. Another assay, termed MeDIP (Weber et al., Nat. Genet., 37:853-62 (2005)), uses bead-immobilized anti-5-methylcytosine antibodies (α5mC-Ab), instead of MBD proteins, to enrich for methylated DNA. However, the use of each these techniques has been limited by one or more of the following: a requirement for relatively large amounts of input genomic DNA, a potential for false-positive results due to capture of unmethylated DNA, incompatibility with high-throughput platforms, and lack of quantitative data.
Thus, there remains a need for methods that detect DNA methylation that are sensitive, accurate, and robust. The ability to multiplex samples, quantify levels of methylation at both the genomic and gene level, as well as the ability to perform analysis in high-throughput formats would be a clear advantage for methylation detection and identification methods used both in the research lab and in the clinic.